Apparatus for monitoring glucose

ABSTRACT

A method for monitoring the glucose level in a body fluid uses an apparatus which includes a conjugate of glucose oxidase with a fluorescent dye coated onto an optical fiber in contact with the body fluid, a source of excitation light and a fluorescence emission detector. Glucose is oxidized by oxygen in the body fluid causing a decrease in oxygen concentration at the enzyme. The fluorescent dye is sensitive to oxygen quenching so that, when the oxygen concentration decreases, fluorescence emission increases in direct proportion to the glucose concentration in the fluid.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to monitoring glucose levels, and moreparticularly relates to an implantable glucose sensor and a method forusing same for detection or quantitation of an elevated glucose level ina body fluid.

2. Background Description

Over five million Americans have diagnosed diabetes and another fivemillion are estimated to have undiagnosed diabetes. Diabetes is achronic metabolic disorder manifested by degenerative disease of theblood vessels, kidneys, retina and nervous system and is characterizedby the body's abnormal metabolism of carbohydrates, proteins and fats.Carbohydrates are normally digested to glucose in the gut, the glucosebeing absorbed into the circulatory system and carried to most cells ofthe body where it is utilized as the principal source of nutrition. Inone form of diabetes, the glucose cannot enter the liver, muscle and fatcells in normal amounts for storage or energy use and as a consequencebuilds up in the blood and urine. Abnormally high blood glucose levelsmay lead to the accumulation of toxic ketone metabolites often leadingto coma and death.

Glucose is normally present in the blood stream at a level of about 0.8to 1.0 mg/ml and is maintained within this narrow range by a continuousmoment to moment sensing and correction of the glucose concentration byhormones released from the pancreas. If glucose concentration in theblood stream rises above the normal range, insulin is released andcauses metabolism of glucose which lowers the concentration. If theglucose concentration falls below the normal range, glucogen is releasedto raise it to normal. The pathological condition of diabetes isprimarily due to a long term hyperglycemia resulting from reducedinsulin production or release.

Many diabetics control their disease merely by diet and weight control.Others require drug treatment, generally insulin or an oral hypoglycemicagent, to control blood glucose levels. Oral administration of insulinis not practical because it is destroyed by proteolytic enzymes in thegastro-intestinal tract. Injected insulin provides only partial controlof the degenerative effects of diabetes, apparently because periodicinjections do not closely correspond to changing metabolic requirementsconsequent to fluctuating blood glucose levels. For this reason, avariety of methods have been proposed for rapid and accurate assessmentof blood glucose levels.

Glucose measurement systems known in the art are generally based on theoxidation of glucose by oxygen in the presence of glucose oxidase. U.S.Pat. Nos. 4,452,887 to Kitajina et al., and 4,390,621 and 4,460,684 toBauer exemplify a chromogenic system in which hydrogen peroxide formedduring the oxidation oxidizes a substrate in the presence of peroxidaseto produce a color which is measured. Conversion of the chemical energyof the oxidation reaction to electrical energy, which is measured atelectrodes, is the subject of U.S. Pat. Nos. 4,392,933 to Nakamura etal., 4,436,094 to Cerami, 4,431,004 to Bessman et al. and 4,317,817 toBusby.

Cerami, in U.S. Pat. No. 4,330,299, discloses an indicator element, suchas a dye, as part of a complex containing a carbohydrate or a lectin.The indicator remains undetected until released from the complex byglucose in direct proportion to the glucose concentration.

Boehringer Mannheim Diagnostics (Indianapolis, Ind.) recently marketedan in vitro enzyme-based blood glucose monitoring system, (Accu-Chek™Chemstrip bG™), which may be read colorimetrically orphoto-electronically.

Fiber optic probes for determination of oxygen pressure in a body fluidhave been described. Peterson et al., in U.S. Pat. No. 4,476,870,disclose an implantable device for measurement of partial oxygenpressure in a blood stream based on oxygen quenching of fluorescence.

U.S. Pat. No. 4,399,099 to Buckles discloses a dual fiber optic deviceuseful in a method for measuring glucose concentration. Oxygen permeablesheaths containing an oxygen quenchable fluorescent dye surround opticalfibers, one of the sheaths containing glucose oxidase. The enzymeoxidizes glucose and thereby lowers oxygen concentration which isdetected by reduced quenching of the fluorescence emission from the dye.

Prior art methods and devices disclosed to date for glucose measurementall suffer from deficiencies such as insufficient accuracy, speed or useof methodology or equipment which is impractical for an implantabledevice. There remains a definite need for a simple and accurate methodfor glucose monitoring using a small, light and compact apparatus. It istoward fulfillment of this need that the present invention is directed.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method to detect, either invivo or in vitro, a glucose level in a body fluid which differs from areference level. A fluorescent dye, the fluorescence emission of whichis sensitive to oxygen quenching so that the emission is maximum in theabsence of oxygen, is conjugated to active glucose oxidase. The dyeconjugated to active glucose oxidase is hereinafter called the test dye.The dye-enzyme conjugate is immobilized in contact with a body fluid,and glucose in the fluid is oxidized at the active enzyme withconsumption of oxygen. The oxygen concentration at the dye is therebyreduced in inverse proportion to the extent of oxidation and thereforealso to the glucose concentration. Application of excitation light tothe dye causes fluorescence emission, which is measured. The magnitudeof the emission is inversely proportional to oxygen concentration at thedye and therefore directly proportional to glucose concentration in thefluid.

The dye may also be conjugated to inactivated glucose oxidase,hereinafter called the control dye This conjugate is also immobilized incontact with the body fluid. Glucose in the fluid is not oxidized by theinactive enzyme and the oxygen concentration at the control dyetherefore remains unchanged. Quenching therefore does not occur, and themagnitude of fluorescence emission from the control dye remainsconstant, irrespective of changing glucose concentration, and provides abase line control for comparison with the magnitude of emission from thetest dye which does fluctuate in proportion to glucose concentration. Ifemission from the test dye is greater than that from the control dye, anelevated glucose concentration in the fluid is indicated.

The method of the invention may also be used to quantitate the glucoseconcentration in the body fluid. In this embodiment of the invention,the magnitude of the fluorescence emission from the test dye may becompared with the magnitude of emission measured when the fluid containsa predetermined quantity of glucose. Emission from a plurality of fluidscontaining predetermined quantities of glucose may be measured toprepare a standard curve which relates glucose concentration in thefluid to the magnitude of fluorescence emission.

Another aspect of the invention is a glucose-monitoring apparatus. Thetwo conjugates having active and inactive enzymes, described above, arecoated onto the surfaces of separate optical fibers adapted forinsertion into the fluid to be tested. The apparatus includes a suitablesource of excitation light and a suitable fluorescence emissiondetector. The excitation light passes through the fibers, excites thedyes and induces fluorescence emission which passes back through thefibers where it is detected by the detector.

The preferred apparatus has four fibers, two for passage of excitationlight from the light source to the dye-conjugates and two for passage offluorescence emission from the dye conjugates to the detector. The mostpreferred apparatus has two pairs of concentric fibers and uses a lightemitting diode (LED) as light source and a photocell as detector. Onepair of fiber is coated with active enzyme-dye conjugate and is furthercoated with a glucose permeable membrane. The dye in this conjugateserves as the test dye. The other pair is also coated with activeenzyme-dye conjugate, but is further coated with an oxygen permeablemembrane which precludes passage of glucose so that the enzyme iseffectively rendered inactive and its dye serves as the control dye. Onefiber in each pair introduces excitation light to the conjugate and theother fiber in each pair conducts fluorescence emission from theconjugate to the detector.

Thus, in accordance with the invention, an elevated glucose level in abody fluid may be detected or quantitated, in vivo or in vitro, by amethod using a glucose monitoring apparatus. The apparatus employsglucose oxidase covalently conjugated to a fluorescent dye whereby thedye and the enzyme are in close proximity so that the local oxygenconcentration at the site of the enzyme reaction can be determined withexceptional accuracy. The apparatus includes an optical fiber which maybe very thin and flexible thereby providing advantages for comfort andsafety when inserted into the body fluid through the skin. The LED andphotocell of the preferred apparatus are small and light and may easilybe assembled into a simple and inexpensive unit to be either implantedor worn externally on the surface of the body, and may, if desired, beused in conjunction with any insulin delivery system. Because of theseand other features, the apparatus of the invention may easily and safelybe used on an outpatient basis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an apparatus of the invention using twooptical fibers;

FIG. 2 is a vertical sectional view of the apparatus of FIG. 1 takenalong line 2--2 thereof;

FIG. 3 is a perspective view of an apparatus of the invention using fouroptical fibers;

FIG. 4 is a perspective view of an apparatus of the invention usingconcentric optical fibers;

FIG. 5 is a horizontal section view of the apparatus of FIG. 4 takenalong line 5--5 thereof; and

FIG. 6 is a perspective view of an apparatus of the invention, similarto the apparatus of FIG. 4, using enzyme conjugates coated withmembranes.

DETAILED DESCRIPTION OF THE INVENTION

While this invention is satisfied by embodiments in many differentforms, there will herein be described in detail preferred embodiments ofthe invention, with the understanding that the present disclosure is tobe considered as exemplary of the principles of the invention and is notintended to limit the invention to the embodiments illustrated anddescribed. The scope of the invention will be measured by the appendedclaims and their equivalents.

The method of the present invention for continuous monitoring of glucosein a body fluid is based on the well-known oxidative conversion ofglucose to gluconic acid catalyzed by glucose oxidase. When glucose isoxidized, the consumption of oxygen causes a decrease in the localoxygen concentration at the active site of the enzyme. This decrease isproportional to glucose concentration and may be detected byfluorescence emission from a dye conjugated to the enzyme. Glucoseoxidase is a well-known and well-characterized enzyme and iscommercially available, for example, from Sigma Chemical Co., St. Louis,Mo.

The dye to be conjugated to the enzyme may be any fluorescent dyesensitive to quenching of its fluorescence emission by oxygen. Such adye fluoresces with maximum intensity in the absence of oxygen, and theintensity of its fluorescence emission is decreased in inverseproportion to the oxygen concentration in the immediate vicinity of thedye. Such dyes preferably are hydrophobic fluorescent dyes having strongabsorbance in the visible part of the spectrum. Exemplary of, but notlimited to, such dyes are those listed in Peterson et al. (op. cit.),preferably perylene dibutyrate, most preferably fluoranthrene.

Conjugation of the dye to the enzyme may be carried out by anyconventional procedure as, for example, by covalently coupling activefunctional groups on the dye and enzyme. The functional groups may bebonded directly, as in amide bond formation between amino and carboxylgroups, or they may be coupled through linking groups which couple, forexample, amino, hydroxyl or sulfhydryl groups on one component to acarboxyl group on the other component. Suitable linking groups may be,for example, but not limited to, a methylene chain of from one to sixcarbon atoms. If desired, the technique of affinity labeling may be usedto conjugate the dye near the active site of the enzyme. The ratio ofdye molecules to enzyme molecules in the conjugate is not critical, butpreferably is as high as possible in order that the emission signal beas intense as possible. The coupling of enzymes and dyes, includingaffinity labeling, is well-known in the art and further details in thisrespect are not necessary for a complete understanding of the invention.

The dye-enzyme conjugate is immobilized on a solid support introducedinto the body fluid in such a way that the enzyme contacts glucose inthe fluid. Excitation light is applied to the dye and fluorescenceemission is detected therefrom. Any support material may be used whichsubstantially does not interact with the fluid or interfere with theoxidation reaction or the fluorescence detection system. Exemplary ofsuch supports are glass and plastics, such as polyethylene, polystyrene,polyvinyl chloride and polytetrafluoroethylene.

A particularly preferred support is an optical fiber which, in additionto providing the support for immobilization of the conjugate, alsoserves as the means for introduction of excitation light to the dye andconduction of fluorescence emission from the dye. Optical fibers act aspipelines for passage of light. They are made of a transparent material,such as glass, and are designed in such a way that very little light canleak out through their sidewalls. A thorough discussion of opticalfibers is given by D.M. Considine et al. in Encyclopedia of Chemistry,Van Nostrand Reinhold, (1984) p 645.

The dye-enzyme conjugate may be coated onto a segment of an opticalfiber to be contacted with the body fluid. Alternatively, the conjugatemay be coated onto a solid support as described above, and the opticalfiber brought into intimate contact with the conjugate on the support insuch a way that light passed through the fiber is absorbed by the dye.Fluorescence emission from the dye passes back through the fiber and itsintensity is measured on a detector.

As mentioned above, fluorescence intensity from the test dye is directlyproportional to glucose concentration in the fluid. In order todetermine whether the intensity of the emission from the test dyeindicates an elevated glucose level in the fluid, a base line level offluorescence emission may be determined, preferably simultaneously, fromthe control dye. A second optical fiber may be coated with dye only, thequantity of dye being substantially the same as conjugated to theenzyme. Passage of excitation light through this second optical fiberexcites the dye to emit fluorescence which is independent of glucoseconcentration and thus a measure of ambient oxygen concentration. If theintensity of the emission from the test dye is greater than that fromthe control dye, an elevated glucose level in the fluid is indicated.

In a preferred embodiment of the method of the invention, the base linelevel of fluorescence emission from the control dye may be obtained witha second dye-enzyme conjugate. The second conjugate may be prepared inthe same way as the first conjugate, except inactive glucose oxidase isused. The enzyme may be rendered inactive, i.e., incapable of catalyzingoxidation of glucose, either prior to or subsequent to coupling to thedye. Methods to inactivate enzymes are routine, well-known to thoseskilled in the art, and do not constitute a part of this invention.

Most preferably, the inactive enzyme-dye conjugate may be prepared bycoating active enzyme-dye conjugate with a membrane. In this embodimentof the method of the invention, active enzyme-dye conjugate immobilizedon a first fiber is coated with a selective membrane permeable tomolecules the size of glucose and smaller. When introduced into the bodyfluid, this membrane allows glucose to pass through and contact theconjugate where it is oxidized. Dye in this conjugate is thus the testdye and fluorescence emission therefrom measures glucose concentration.Conjugate on a second fiber is coated with a selective membranepermeable only to molecules the size of oxygen and smaller. Sinceglucose molecules are larger than oxygen molecules, glucose in the bodyfluid cannot reach this conjugate to be oxidized, so that its enzymehas, in effect, been inactivated. Oxygen, however, can reach theconjugate and thus provide measurement of ambient oxygen concentration.The dye in this conjugate is thus the control dye, and comparison of theintensities of fluorescence emission from the two dyes indicates, asdescribed above, whether the fluid contains an elevated glucose level.

The method of the present invention may be adapted to quantitate theglucose concentration in a body fluid. In this embodiment of theinvention, the intensity of fluorescence emission from the test dye isdetermined and compared to the intensity of emission determined when themethod of the invention is applied to a body fluid having apredetermined glucose concentration. For this embodiment, the inventioncontemplates a standard curve which relates fluorescence emissionintensity, as determined with the device of the invention, to glucoseconcentration. In accordance with this embodiment of the method, glucoseconcentration, for example in a diabetic's blood stream, may beascertained merely by finding the test dye fluorescence intensity on thestandard curve and reading the corresponding glucose concentration.

Having now described the method of the invention, various embodiments ofthe blood glucose monitoring apparatus of the invention will bedescribed with the aid of the figures. FIG. 1 shows glucose monitor 10having optical fibers 12 and 14, each having a sidewall portion 16 and17, respectively, and a bottom portion 18 and 19, respectively. Bottomportion 18 of optical fiber 12 has a coating of conjugate 20 of activeglucose oxidase conjugated to fluorescent dye (the test dye). Bottomportion 19 of optical fiber 14 has a coating of conjugate 21 of inactiveglucose oxidase conjugated to fluorescent dye (the control dye).Alternatively, reference numeral 21, representing the control dye, maybe unconjugated fluorescent dye, i.e., the enzyme is omitted. Arrows 22diagrammatically illustrate excitation light passing down fibers 12 and14 from a light source (not shown) where it is absorbed by the dyes andemitted therefrom as fluorescence emission 24. Emission 24 returns upfibers 12 and 14 and is measured by a detector (not shown).

FIG. 2 is a vertical sectional view of the apparatus of FIG. 1 afterinsertion into body fluid 26, illustrated in the Figure as a bloodstream. Optical fibers 12 and 14 are shown surrounded by claddingmaterial 27, which separates the fibers and prevents leakage of lightthrough the sidewall portions 16 and 17. Any cladding materialconventional in optical fiber technology, such as plastic or glasshaving a refractive index lower than that of the light-transmissive coreof the fiber, may be used.

An embodiment of the apparatus in which two fibers are used for each ofthe test dyes and the control dye is shown in FIG. 3. In FIGS. 3-6,elements identical to elements described in FIGS. 1 and 2 are given thesame reference numbers and elements similar are given the same basereference number followed by a different suffix (letter).

In FIG. 3, a solid support, shown in the form of a disc 28, is coated onits upper surface 30 with active glucose oxidase-fluorescent dyeconjugate 20. Disc 28a is coated on its upper surface 30a with inactiveglucose oxidase-fluorescent dye conjugate 21. Discs 28 and 28apreferably are made of a porous plastic material such as polystyrenefoam through which the body fluid may freely pass. Optical fibers12a,12b,14a and 14b are attached to discs 28 and 28a so that theirbottom portions 18 and 19 are in intimate contact with conjugatecoatings 20 and 21, respectively. Excitation light 22 from the lightsource passes down fibers 12a and 14a and contacts conjugates 20 and 21in contact with body fluid 26 in porous discs 28 and 28a where it isabsorbed by the fluorescent dye and emitted as fluorescence emission 24.Emission 24 passes up through fibers 12b and 14b to the detector (notshown).

FIG. 4 gives a perspective view of an embodiment of the apparatus usingconcentric fibers. Active enzyme-dye conjugate 20 and inactiveenzyme-dye conjugate 21 are coated onto upper surfaces 30 and 30a ofporous solid supports 28 and 28a, respectively. Optical fibers 12a and14a have dimensions which allow them to fit inside of hollow opticalfibers 32 and 32a. Fibers 12a and 14a and 32 and 32a are separated bylayers of cladding material 27, as shown in horizontal sectional FIG. 5.

FIG. 6 shows an embodiment of the apparatus in which the conjugates arecoated with membranes. Concentric optical fibers 12a and 32 are coatedon their bottom surfaces 30 with active enzyme-dye conjugate 20a. Thecoating of conjugate 20a is then itself coated with membrane 34 which ispermeable to glucose and molecules smaller than glucose so that its dyeserves as the test dye. Concentric optical fibers 14a and 32a arelikewise coated on their bottom surfaces 30a with active conjugate 20,which is further coated with membrane 36 permeable only to molecules thesize of oxygen and smaller so that its dye serves as the control dye.

It is understood that support discs 28 and 28a, depicted in FIG. 4, maybe included in the apparatuses of FIGS. 1 or 6. Likewise, membranes 34and 36 of FIG. 6 may be included in any of the other embodimentsdescribed. The invention is contemplated to encompass these and anyother modifications of the apparatus, which provide glucose monitoringin accordance with the principles of the method of the invention hereindescribed.

In summary, the invention provides a system including a method and anapparatus for monitoring of glucose in a body fluid, preferably on acontinuous basis. The method is based on oxidation of glucose by glucoseoxidase, the extent of oxidation being proportional to glucoseconcentration. The oxidation reaction depletes oxygen at the active siteof the enzyme, and the reduced oxygen concentration is detected andmeasured by changes in fluorescence intensity proportional to the oxygenconcentration. The system may be used either in vitro or in vivo and isparticularly suitable for blood glucose determinations. When used invivo for monitoring glucose concentration in a diabetic's blood stream,the system may be used in conjunction with any insulin delivery system,and is easily adapted for out patient use.

What is claimed is:
 1. An apparatus for monitoring glucose concentrationin a body fluid comprising:(a) a first cable comprising first and secondoptical fibers having immobilized thereon a first conjugate comprisingactive glucose oxidase and a fluorescent dye, emission from said dyebeing sensitive to oxygen quenching; (b) a second cable comprising thirdand fourth optical fibers having immobilized thereon a second conjugatecomprising inactive glucose oxidase and the same aforementionedfluorescent dye; (c) a light source in excitation light transmissivecommunication with said first conjugate through said first fiber andwith said second conjugate through said third fiber; and (d) a detectorin fluorescence emission transmissive communication with said firstconjugate through said second fiber and with said second conjugatethrough said fourth fiber.
 2. The apparatus of claim 1 wherein said dyeis selected from the group of dyes consisting of perylene dibutyrate andfluoranthrene.
 3. The apparatus of claim 1 wherein said active andinactive glucose oxidase are conjugated to said dye by a covalent bond.4. The apparatus of claim 3 wherein said active and inactive glucoseoxidase are conjugated to said dye through a linking group.
 5. Theapparatus of claim 1 wherein said light source is a light emittingdiode.
 6. The apparatus of claim 1 wherein said detector is a photocell.7. An apparatus for monitoring glucose concentration in a body fluidcomprising:(a) first light transmissive means having positioned adjacentthereto a first material comprising a conjugate of active glucoseoxidase and a fluorescent dye, emission from said dye being sensitive tooxygen quenching; (b) second light transmissive means having positionedadjacent thereto a second material comprising the same aforementionedfluorescent dye conjugated to inactive glucose oxidase; (c) means forproviding excitation light to said first material through said firstlight transmissive means and to said second material through said secondlight transmissive means; and (d) means for detecting fluorescenceemission from said first material through said first light transmissivemeans and from said second material through said second lighttransmissive means.
 8. An apparatus for monitoring glucose concentrationin a body fluid comprising:(a) a first cable comprising first and secondoptical fibers having immobilized thereon a first conjugate comprisingglucose oxidase covalently conjugated to a fluorescent dye, emissionfrom said dye being sensitive to oxygen quenching, said first conjugatebeing coated with a membrane permeable to glucose; (b) a second cablecomprising third and fourth optical fibers having immobilized thereon asecond conjugate comprising glucose oxidase covalently conjugated to thesame fluorescent dye, said second conjugate being coated with a membranepermeable to oxygen but impermeable to glucose; (c) a light emittingdiode in excitation light transmissive communication with said firstconjugate through said first fiber and with said second conjugatethrough said third fiber; and (d) a photocell in fluorescence emissiontransmissive communication with said first conjugate through said secondfiber and with said second conjugate through said fourth fiber.
 9. Theapparatus of claim 7 wherein said first material is coated on said firstlight transmissive means and said second material is coated on saidsecond light transmissive means.
 10. The apparatus of claim 7 furthercomprising a first solid support adjacent said first light transmissivemeans and having said first material coated thereon and a second solidsupport adjacent said second light transmissive means and having saidsecond material coated thereon.